Tumors are exposed to constantly changing nutrient environments which creates metabolic stress that impairs their growth and proliferation. Consequently, cancer cells evolve phenotypic adaptations to contend with this nutrient flux that require epigenetic and transcriptional reprogramming. These phenotypic adaptations also can affect tumor cell sensitivity to chemotherapeutic agents. How cytoplasmic organelles involved in nutrient signaling exert their effects on the nuclear epigenome remains a fundamental problem to define since these pathways are candidate therapeutic targets for anticancer drug development. The V-ATPase is an evolutionarily conserved multimeric proton pump that acidifies the endolysosomal compartment to regulate intracellular pH and activate essential nutrient signaling pathways. Na+/H+ exchanger (NHE) factors, specifically NHE9, antagonize V-ATPase dependent endosomal acidification. NHE9 is overexpressed in many tumors, and recent studies have demonstrated that NHE9 overexpression in glioblastoma (GBM) contributes directly to tumorigenesis. Intriguingly, genetic screens in budding yeast identified both the V-ATPase and the yeast NHE9 ortholog, Nhx1, as regulators of histone H3 and H4 (H3/H4) acetylation through unknown mechanisms. Our preliminary data demonstrate that V-ATPase signaling in human GBM cells also regulates H3/H4 acetylation, thus demonstrating that the V-ATPase is an evolutionarily conserved epigenetic regulator. This project will test the innovative hypothesis that the V-ATPase and Nhx1/NHE9 regulate the endolysosomal- nuclear communication required for epigenetic adaptation to altered nutrient states. Using yeast and human GBM models, we will perform the following Specific Aims. In Aim I, we will identify the key nutrient signaling pathways downstream of the V-ATPase controlling global H3/H4 acetylation, and then we will determine if this involves repression of the sirtuin family of deacetylases or if it involves non-sirtuin mechanisms. We will test specifically if V-ATPase dependent H3/H4 acetylation regulates the binding of ATP-dependent chromatin remodeling enzymes important for transcription of metabolic genes. In Aim II, we will determine if V-ATPase dependent H3/H4 acetylation controls the DNA damage response to alkylating agents, including the GBM standard of care chemotherapeutic agent temozolomide (TMZ), by anchoring ATP-dependent nucleosome remodeling enzymes onto chromatin. Upon the project's completion, we will have defined a novel paradigm by which the endolysosomal compartment regulates nuclear epigenetic pathways critical for metabolic gene expression and sensitivity to genotoxic stress.